Re: [ccp4bb] str solving problem
Pramod: [1] Please refrain from posting excessively large (1MB) attachments to the ccp4bb. Either use a compression technique or use another means of transmitting large files to your recipients without spamming the entire group. [2] Your predictions are not overlaying well with the spots . Well it may be overlaying well with a subset of spots (in this case, the strongest spots that obey the symmetry *you* chose), but your are leaving out a large number of spots. As I said before, your diffraction pattern is exhibiting strong reflections with weak reflections at a given resolution. It looks as if there are multiple lattices probably due to bad crystal morphology ( a cracked crystal, twinning, pseudosymmetry) or multiple crystals in the beam, the list goes on I've experienced your case many, many times you'll get a good MR solution, but then the refinement becomes 'stuck'. I think the idiom... 'garbage in, garbage out' applies here.. [3] I have given several efforts to the XDS but its giving error data image of particular no. does not exist (initially it was saying 11th image than i change image range then it says 21st and so on) kindly check my data collection profile and XDS.INP file in attachment' Sounds like a file name problem. [4] Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. raising the crystals and struggle is on the peak... There are a number of ccp4bb postings about crystal reproducibility or crystal diffraction improvement. Search the archives for these. F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] PILATUS data collection
Graeme and Bob, Wow... It's great to learn from actual experiences. Thanks much for this write up. If this were stackoverflow, +1. F On May 8, 2013, at 12:32 AM, Graeme Winter graeme.win...@gmail.com wrote: A couple of extra comments on top of Bob's rather comprehensive recommendations, based purely on actually looking at Pilatus data (I mean *looking*) When you are inspecting the images looking at them at 100% size is important: spots are small relative to pixels and the point spread is essentially zero. I also find it helpful to look at white spots on a black background rather than the reverse. The DECTRIS folks have an image viewer named ALBULA which works well, however ADXV also works fine (IMHO) if you tweak the settings as above. It is remarkable how big a difference it makes. The other big difference in terms of viewing the images is that (if you have low counts) you are actually at the mercy of real Poisson statistics. For example, if you have a spot (pixel) with 8 counts in against a background of 0 - 3 counts (say) it looks nice and clear - certainly based on experience of CCD images. And the spot is say four times the background so it must be good right? However the variance on a spot with 8 counts is 8, the variance from background subtraction may be about 3 so your spot has a maximum I/sigma of ~ 8 / sqrt(11) so about 2 and a bit. On the flip side, data from a decent crystal taken with a low dose can look almost blank at first glance (esp. with black spots on white; zoomed out) but process very nicely. Take some time to get used to the instrument. A couple of final comments: The machine has no read-out noise so fine phi slicing (and dose slicing) can only help - recording twice as many degrees with half as much dose increases the chance of getting a complete and relatively undamaged data set. All it does is take up lots of disk space. There is really no risk in doing this, unlike with a CCD where you are at war with the read-out noise. The machine also behaves completely differently to a CCD: this takes some getting used to. Take narrow oscillations as this will give better measurements of strong reflections (which I think is detailed in the papers Bob recommended.) Also *take your time* - one of the great things about these detectors is that they allow you to do continuous exposures, which can essentially double the throughput or more of data collection. Take back some of that time to use a lower dose rate and spread your photons out more evenly across reciprocal space. You can always measure more data if your sample is undamaged. Best wishes, Graeme On 7 May 2013 02:00, Robert Sweet rsw...@bnl.gov wrote: The seminal paper on actually how to collect data from detectors like this and others with negligible read-out time is this one, which I strongly recommend: Optimal Fine phi-slicing for Single-Photon-Counting Pixel Detectors Marcus Mueller, Meitian Wang, and Clemens Schulze-Briese, Acta Cryst.(2012) D68, 42-56 And you can pick up a copy of the paper from the RapiData web site: http://www.px.nsls.bnl.gov/courses/papers/Mueller_ACD68_2012.pdf The classic paper on data-collection strategies is this: Data-Collection Stragegies, Dauter, Z. Acta Cryst. (1999). D55, 1703-1717. Also available from the RapiData site: http://www.px.nsls.bnl.gov/courses/papers/dauter_strategy.pdf Then there are multiple papers on damage and its impact on your data. I suggest this one: Radiation damage in macromolecular crystallography: what is it and why should we care?, E.Garman, Acta Cryst. D66, 339-351(2010). which you can find here: http://www.px.nsls.bnl.gov/courses/papers/actad-garman-2010.pdf With this under your belt you'll be able to decide how to collect your phasing data. The bottom line is probably that you should go for SAD data. Employ multiple crystals and average them together in a judicious way, keeping only the sweeps from barely damaged x-tals. Good luck, Bob Sweet = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Photon Sciences and Biosciences Dept Office and mail, Bldg 745, a.k.a. LOB-5 Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) = On Mon, 6 May 2013, Theresa Hsu wrote: Dear crystallographers Is there a good source/review/software to obtain tips for good data collection strategy using PILATUS detectors at synchrotron? Do we need to collect sweeps of high and low resolution data
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Wow, Usually on the default settings of adxv, I can see spots at this I/sig! I suspect your data goes higher than 1.77. Include more of the high resolution data. You very likely don't have the correct space group. F On Mar 15, 2013, at 11:39 AM, Andrey Nascimento andreynascime...@gmail.com wrote: I/Isig.=4.4 - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] a challenge
Ok, I'll bite. I dare anyone who considers themself an expert macromolecular crystallographer to find a way to build out of this map. I put emphasis on this map. Short of actually cheating (see below), there doesn't seem to be any automated way to arrive at a solved structure from these phases I put emphasis on these phases. I think the real challenge (and one that makes for an excellent macromolecular crystallographer) is how well one can interpret a map with poor phases. That being said, I think a recalculation of the map using any other information besides the map itself should not be allowed. PS. I'd like to see what the pre-DM phases look like. There's a huge chunk of the protein that is completely flattened out in impossible.mtz . F On Jan 12, 2013, at 1:50 PM, James Holton jmhol...@lbl.gov wrote: Woops! sorry folks. I made a mistake with the I(+)/I(-) entry. They had the wrong axis convention relative to 3dko and the F in the same file. Sorry about that. The files on the website now should be right. http://bl831.als.lbl.gov/~jamesh/challenge/possible.mtz http://bl831.als.lbl.gov/~jamesh/challenge/impossible.mtz md5 sums: c4bdb32a08c884884229e8080228d166 impossible.mtz caf05437132841b595be1c0dc1151123 possible.mtz -James Holton MAD Scientist On 1/12/2013 8:25 AM, James Holton wrote: Fair enough! I have just now added DANO and I(+)/I(-) to the files. I'll be very interested to see what you can come up with! For the record, the phases therein came from running mlphare with default parameters but exactly the correct heavy-atom constellation (all the sulfur atoms in 3dko), and then running dm with default parameters. Yes, there are other ways to run mlphare and dm that give better phases, but I was only able to determine those parameters by cheating (comparing the resulting map to the right answer), so I don't think it is fair to use those maps. I have had a few questions about what is cheating and what is not cheating. I don't have a problem with the use of sequence information because that actually is something that you realistically would know about your protein when you sat down to collect data. The sequence of this molecule is that of 3dko: http://bl831.als.lbl.gov/~jamesh/challenge/seq.pir I also don't have a problem with anyone actually using an automation program to _help_ them solve the impossible dataset as long as they can explain what they did. Simply putting the above sequence into BALBES would, of course, be cheating! I suppose one could try eliminating 3dko and its homologs from the BALBES search, but that, in and of itself, is perhaps relevant to the challenge: what is the most distance homolog that still allows you to solve the structure?. That, I think, is also a stringent test of model-building skill. I have already tried ARP/wARP, phenix.autobuild and buccaneer/refmac. With default parameters, all of these programs fail on both the possible and impossible datasets. It was only with some substantial tweaking that I found a way to get phenix.autobuild to crack the possible dataset (using 20 models in parallel). I have not yet found a way to get any automation program to build its way out of the impossible dataset. Personally, I think that the breakthrough might be something like what Tom Terwilliger mentioned. If you build a good enough starting set of atoms, then I think an automation program should be able to take you the rest of the way. If that is the case, then it means people like Tom who develop such programs for us might be able to use that insight to improve the software, and that is something that will benefit all of us. Or, it is entirely possible that I'm just not running the current software properly! If so, I'd love it if someone who knows better (such as their developers) could enlighten me. -James Holton MAD Scientist On 1/12/2013 3:07 AM, Pavol Skubak wrote: Dear James, your challenge in its current form ignores an important source of information for model building that is available for your simulated data - namely, it does not allow to use anomalous phase information in the model building. In difficult cases on the edge of success such as this one, this typically makes the difference between building and not building. If you can make the F+/F- and Se substructure available, we can test whether this is the case indeed. However, while I expect this would push the challenge further significantly, most likely you would be able to decrease the Se incorporation of your simulated data further to such levels that the anomalous signal is again no longer sufficient to build the structure. And most likely, there would again exist an edge where a small decrease in the Se incorporation would lead from a model built to no model built. Best regards, -- Pavol Skubak Biophysical
Re: [ccp4bb] NCS from electron density
I've had good experience with GETAX if you have a self rotation peak. Be careful about moving the NCS operator from program to program. Phenix/DM/RAVE all have specific formats for how it should be presented. F On Dec 12, 2012, at 10:01 AM, Nicolas Soler wrote: Dear all, Is there a pipeline that will find NCS operators from a map, select the relevant ones and apply them all in NCS averaging density modification? Otherwise, what would be the best way to proceed ? (I have a P21 spacegroup so I wonder what happens with the origin when I pass the NCS operators from one program to another). Thanks for your help, Nicolas - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] NCS from electron density
Each case is specific, but when I was learning, I found Phil's Notes on Averaging (http://xray0.princeton.edu/~phil/Facility/averaging.html) to be quite useful. F On Dec 12, 2012, at 11:59 AM, Scott Thomas Walsh wrote: Hi Francis, Do you have a specific how to on your experience with NCS (reference)? Cheers, Scott Scott T. R. Walsh, PhD Assistant Professor University of Maryland IBBR/CBMG 3127E CARB-2 9600 Gudelsky Drive Rockville, MD 20850 USA phone: (240) 314-6478 fax: (240) 314-6225 email: swals...@umd.edu On Dec 12, 2012, at 2:55 PM, Francis E Reyes wrote: I've had good experience with GETAX if you have a self rotation peak. Be careful about moving the NCS operator from program to program. Phenix/DM/RAVE all have specific formats for how it should be presented. F On Dec 12, 2012, at 10:01 AM, Nicolas Soler wrote: Dear all, Is there a pipeline that will find NCS operators from a map, select the relevant ones and apply them all in NCS averaging density modification? Otherwise, what would be the best way to proceed ? (I have a P21 spacegroup so I wonder what happens with the origin when I pass the NCS operators from one program to another). Thanks for your help, Nicolas - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Nobel Prize 2012
Had the fortunate opportunity to hear him speak about the crystallography at the Pittsburgh Diffraction Conference last week. Great work and well done! Francis On Oct 10, 2012, at 8:54 AM, Bosch, Juergen wrote: Yes agreed great work - more of that please. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Oct 10, 2012, at 7:20, Toufic El Arnaout tou...@inmesolabs.net wrote: Congratulations to Robert Lefkowitz and Brian Kobilka: Nobel Prize in Chemistry, 2012! Congrats to the protein structure community. toufic el arnaout - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] How to output water molecules with Phaser?
Hi Randy When you say carried along are you saying that they are used as part of the molecular replacement search? Or are they temporarily put aside and them simply added to the PDB in frame with the molecular replacement solution (but are not part of the weighted structure factors output in the final mtz)? F On Oct 2, 2012, at 7:55 AM, Randy Read rj...@cam.ac.uk wrote: As a deliberate choice, we changed Phaser so that it would omit all HETATM records from the model used for molecular replacement. This was largely because when extensive water structure was carried along, it could mess up the molecular replacement calculation. However, if you want something carried along as part of the model, you should be able to do that simply by changing HETATM to ATOM in the lines of the atoms in the PDB file that you want to keep. I'd suggest being selective in doing this and making sure you're only including the ones you're deliberately choosing. The very latest version of Phaser is a bit more clever. One side effect is that Se-Met residues were left out of the models, but now Phaser recognises the codes of some modified amino acids and carries them along. Best wishes, Randy Read On 2 Oct 2012, at 15:32, Koji Yonekura wrote: Hi all, I am using Phaser 2.5.1 for molecular replacement. I like to keep tightly bound water molecules (HOH) in the input pdb file. Phaser 2.1.4 outputs all HOH lines in the input pdb file to the output pdb file, but Phaser 2.5.1 doesnot. I am wondering if there is any way to carry input HOH lines to the output file with Phaser 2.5.1. Best, Koji -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Tricky MR problem
[1] In addition to using lower resolution data, you should check a variety of RMSDs as well. In fact constructing an RMSD / high resolution contour plot would be beneficial to determine the optimum set of parameters. (That is if you're using PHASER). [2] You say you suspect two molecules per asymmetric unit. Are you fortunate enough to have NCS that's not aligned with the crystal axes? (Have you checked the self rotation patterson?). If there's significant off origin peaks you can (a) try to model the dimer and use that for the search or (b) use the locked rotation function in molrep. [3] Any heavy atoms in the structure (Zn, FeS, etc)? F On Oct 1, 2012, at 12:26 PM, RHYS GRINTER wrote: Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent).
Re: [ccp4bb] dmmulti cross-crystal averaging question
The domain common to both crystals constitutes the averaging mask. (you can make it around the pdb using ncsmask). As for describing it to dmmulti, you need the rotation matrix which maps it from crystal 1 to crystal 2. The rotation matrix for crystal one is always the identity matrix. You will put the matrix mapping the domain from 1 to 2 as the rotation matrix for the second crystal. You may want to obtain a rotation matrix optimized against your current density first before supplying it to dmmulti (the rave utilities will do this for you). When you get to dmmulti, output the solvent masks and make sure they're accurate (or alternatively you can supply a solvent mask for each crystal separately). F On Sep 21, 2012, at 9:47 AM, Garboczi, David (NIH/NIAID) [E] wrote: We are working with two crystals. Both crystals have a domain that is not in the other. Both crystals have one domain that is the same in each. We'd like the average the one domain between the crystals, but leave the other domains unaveraged. How do we describe that to dmmulti? thanks, Dave -- - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] DM with twinned data
With DM errors, it would be best to supply the input you used for DM as well as DM's complete error output. On Sep 2, 2012, at 9:59 PM, Sofia Caria wrote: Dear all, Recently I have been playing with some twinned data (Britton alpha 0.447). Phasing was done by MR and since I have NCS I was planning to use DM to remove model bias. However the program fails every time and complains of the NCS matrix (determined using Superpose). Can I use DM if I have twinned data? Or is there another reason why the program is failing. Thank you in advance for the help. Best, Sofia -- Sofia Caria Ph.D Kvansakul Laboratory, School of Molecular Sciences, Room 202, Level 2, Physical Sciences 4 Building, La Trobe University, Bundoora, VIC 3086 Australia email: s.ca...@latrobe.edu.au Phone: +613 9479 2263 Fax: +613 9479 1266
[ccp4bb] CNS compliant addition of hydrogens to structure?
Anyone know of a util that'll add hydrogens in a naming scheme consistent with CNS? (reduce doesn't do this). Thanks! F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Large unit cell, overlaps
The cell predictions look like they're overlapping but the spots are not. At first glance it looks like the unit cell is incorrect and is too large. You seem to have intense spots mixed in with weak spots at the same resolution. Smells like multiple unit cells / cracked crystal (which if close together would confuse the autoindex into thinking it's a larger unit cell. . Difficult to tell without seeing the images. The data/spots (not the predicted spots) show reasonable separation. How does the unit cell of the derivative compare with the native? F On Jul 16, 2012, at 2:52 PM, Jason Busby wrote: Hi, I have a crystal with a large unit cell in P21221 (a=134 b=151 c=276) and I'm wondering if I have a problem with overlaps. I have a native dataset, and am trying to get phases. I've collected a Pt soak data set on our home source with a 0.5˚ oscillation angle, but the anomalous signal drops off after about 8Å. I am wondering if this is a problem due to overlaps at higher resolution. The Pt dataset has been integrated with XDS, and there don't seem to be too many rejects, but looking at FRAME.CBF it looks like the predicted spots are overlapping at higher resolution. You can see a zoomed-in part of FRAME.CBF here: http://imgur.com/1WShV Should I be concerned with this? The crystal mosaicity from XDS is 0.25, so fairly low. What can I do about this, should I try smaller oscillation angles? Thanks, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414 - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Large unit cell, overlaps
From the statistics you posted, it seems like the integration went quite reasonably. There is a slight undercompleteness in the high resolution bin (82% is a bit on the low end but since this is for phasing I'd expect a traceable map in light of this). Do the diffraction images indicate very strong (say I/sd 4) spots beyond 3A? If so then it seems that they're being rejected in the final analysis, possibly due to overlaps. If so, I would certainly recollect this data to obtain the higher resolution spots for refinement (not necessarily for phasing). Depending on how long you've been at this, I'd be eager to solve the structure first :) Go with a synchrotron source, Pt anomalous peak is nearer to 1.1A than 1.54A. Except with the iodide data. Did that have a great anomalous signal? F On Jul 16, 2012, at 8:35 PM, Jason Busby wrote: Hi, This is what I thought when collecting the data - the spots did not look to be overlapping. I have actually got 4 datasets (native, mercury, iodide and platinum soaks) and they all index as the same spacegroup and unit cell (the Pt soak being slightly larger unit cell). This is of a large heterodimer, and this unit cell would put 2 in the ASU and a solvent content of 56%, so this seems reasonable. Mosflm also picks the same unit cell, and the predictions seem to match up with spots (although mosflm predicts lots of overlaps) Cheers, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414 On 17/07/2012, at 2:53 PM, Francis E Reyes wrote: The cell predictions look like they're overlapping but the spots are not. At first glance it looks like the unit cell is incorrect and is too large. You seem to have intense spots mixed in with weak spots at the same resolution. Smells like multiple unit cells / cracked crystal (which if close together would confuse the autoindex into thinking it's a larger unit cell. . Difficult to tell without seeing the images. The data/spots (not the predicted spots) show reasonable separation. How does the unit cell of the derivative compare with the native? F On Jul 16, 2012, at 2:52 PM, Jason Busby wrote: Hi, I have a crystal with a large unit cell in P21221 (a=134 b=151 c=276) and I'm wondering if I have a problem with overlaps. I have a native dataset, and am trying to get phases. I've collected a Pt soak data set on our home source with a 0.5˚ oscillation angle, but the anomalous signal drops off after about 8Å. I am wondering if this is a problem due to overlaps at higher resolution. The Pt dataset has been integrated with XDS, and there don't seem to be too many rejects, but looking at FRAME.CBF it looks like the predicted spots are overlapping at higher resolution. You can see a zoomed-in part of FRAME.CBF here: http://imgur.com/1WShV Should I be concerned with this? The crystal mosaicity from XDS is 0.25, so fairly low. What can I do about this, should I try smaller oscillation angles? Thanks, Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414 - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] how to get phase of huge complex
Do you have crystals? Do they diffract? If so, to what resolution? What resolution do you require to answer your biological question? F On Jun 12, 2012, at 7:46 PM, LISA science...@gmail.com wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
Re: [ccp4bb] DNA/RNA modeling
For the RNA I recommend Eric Westhof's Assemble. F On May 23, 2012, at 9:32 AM, jens j birktoft wrote: Hi everybody, Does anyone know of a (non-commercial) software that are suitable for modeling DNA/RNA structures. Coot is great, however I am looking something that allows more flexibility Thanks -- Jens J. Birktoft e-mail: jens.birkt...@nyu.edu slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025 Phone: 212-749-5057
Re: [ccp4bb] phaser: high z score but no sol
P21.. You sure about this space group? (very high confidences for space group and laue group in pointless?) F On Apr 19, 2012, at 12:20 AM, LISA wrote: Hi all, I am trying to solve one structure by molecular replacement with phaser in CCP4. This a complex of a multi-domain domains with small ligand. I have structues of this protein in apo state and with other similar ligand. The space group of this crystal is P21. This crystal should have 4 molecules in ASU. I used the full protein as model but did get any sol and LLG is below zero. Then each domain were used as the search models in phaser with rotation and tranlsation. I can the get high z score (20), and LLG is raising. It looks like I get the right sol, but it have more 50 clashes. Why phaser give wrong sol with so high z socre? Can anyone give me some suggestion to solve my strucutes? Thank you. Best Lisa
[ccp4bb] Disorder or poor phases?
Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would love people's thoughts on this one, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Disorder or poor phases?
Dale Thank you for the case study. I will certainly remember it when I next see: I don't see density for these atoms therefore they must be disordered. You do mention though, that when you were able to assign the sequence to the beta sheets, that the loop regions became clear. I consider the case (which a majority of cases seem to be), where the author has built and sequence assigned 95% of the ASU, but is unable to model a loop region. One possibility is that the loop is truly disordered (95% of the ASU is built and is presumably right), the other possibility is that there's an inherent error in the existing structure that is affecting the interpretation of the loop region. The errors are probably extremely subtle and distributed throughout the model (think of the improvements DEN refinement gave for the rerefinement of p97). I guess in either case, because of the dependency of the map on the existing set of phases it's difficult to determine whether it's truly disordered or not. P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. Tim, I assume a sigmaA weighted 2Fo-Fc map (which I believe is the default for most crystallographic refinement packages). F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] mtz2cif capable of handling map coefficients
It seems that deposition of map coefficients is a good idea. Does someone have an mtz2cif that can handle this? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]
I'm now preparing for the flood of 'unsubscribe ccp4bb' requests On Apr 2, 2012, at 9:15 AM, Bernhard Rupp (Hofkristallrat a.D.) wrote: Guys, http://www.youtube.com/watch?v=CobZuaPMQHw second 9 in this 22 sec video -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerard DVD Kleywegt Sent: Monday, April 02, 2012 8:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication] Dear Manfred, Outside Germany, such excursions are called humour. If you are interested, here is the Wikipedia page for it: http://en.wikipedia.org/wiki/Humour --Gerard
Re: [ccp4bb] about heavy atom derivatization
To add to Ed's comments: Find someone in the immediate department/area to walk you through your first structure. But to answer your question: What about the MR didn't work? Use your MR model to find your heavy atoms. They can be found using anomalous data difference fouriers (or even better log likelihood gradient maps) using data as low as 6-8A, and, in my case, using a model that was about 50% (mass) of the ASU with the placement of that 50% to be about 80-90% correct. F On Mar 30, 2012, at 7:34 AM, Shanti Pal Gangwar wrote: Dear all I am beginner in crystallography.We have collected a native data of a given protein at 2.2A resolution but are unable t solve by MR therefore we collected Hg, and Pt derivative after soaking the crystals. I don't know how to process this heavy atom derivative data and find the anomalous signal if heavy atom is there? thanks in advance for help and suggestions.. regards Shanti Pal Gangwar - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Defining beamstop and error during indexing- moslfm
What resolution are you working at? What are the unit cell dimensions? On Mar 22, 2012, at 11:10 AM, sonali dhindwal wrote: Dear Andrew and all the people for their help, I am providing mosflm the right beamstop and now, I am able to do the indexing, refinement and indexing too. Then I run scala for the output mtz file and it shows Rmerge too high 0.58 What's the space group? Rmerge is misleading in high symmetry space groups. and also when examining the spots and predictions in the image, it seems like it is not picking a lot of spots, so missing large number of reflections. Perhaps a screen shot of one of the images where this is happening would be helpful. So we can judge the quality of the diffraction / whether the space group is correct, etc. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
http://skuld.bmsc.washington.edu/scatter/AS_form.html Maybe useful to you. However, I would advise to do a fluorescence scan over the range given in the graph and then use chooch to provide the precise energies for your peak and inflection. If you have a large crystal don't expose all of it when you do the fluorescence scan but rather reserve a 'fresh' piece to do your actual data collection. F On Mar 6, 2012, at 1:09 PM, Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
Re: [ccp4bb] sudden drop in R/Rfree
I've found the following article to be useful in defining suitable refinement strategies at a particular resolution. 1. Mueller, M., Jenni, S. Ban, N. Strategies for crystallization and structure determination of very large macromolecular assemblies. Curr Opin Struct Biol 17, 572–579 (2007). It can be used as a rough guide line at the start of your refinement (incomplete model, fresh off of molecular replacement) with slow increase of refinement parameters the better your model becomes (and as Rfree permits). F On Mar 2, 2012, at 9:39 AM, Ethan Merritt wrote: On Friday, 02 March 2012, Regina Kettering wrote: Rajesh; I am not sure that you have a high enough data:refinement parameters ratio to refine TLS. It just adds more parameters to refine that can lead to over-refinement of your model, especially at the 3.3 A. I'm afraid you've got this completely backwards. TLS uses very few parameters, and is especially useful at low resolution. At 3.3A I would recommend trying a TLS model _instead_ of refining individual B factors. NCS restraints also help a lot at low resolution. So the drop is believable, but... You should first worry about lots of waters were placed. It's there that many extra parameters have been added, perhaps leading to over-fitting. I would not expect 3.3A data to justify placement of more than a handful of waters at most. If you're parameter counting, you might note that 5 water molecules add more parameters than 1 TLS model. But the TLS model may improve the model everywhere, whereas the waters will only suppress a few local difference density peaks. cheers, Ethan HTH, Regina From: Rajesh kumar ccp4...@hotmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, March 2, 2012 10:54 AM Subject: [ccp4bb] sudden drop in R/Rfree Dear All, I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type structure of same protein at 2.3 A. After molecular replacement, first three rounds of refinement the R/Rf was 26/32.8, 27.1/31.72 % and 7.35/30.88 % respectively. In the fourth round I refined with TLS and NCS abd added water and the R/Rf dropped to 19.34/26.46. It has almost 7% difference. I also see lot of unanswerable density in the map where lot of waters were placed. Model fits to the map like a low resolution data with most of side chains don't have best density. I was not expecting such a sudden drop in the R/Rfree and a difference is 7.2%. I am wondering if I am in right direction. I am not sure if this usual for 3.3A data or in general any data if we consider the difference. I appreciate your valuable suggestions. Thanks Raj - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] effects of salt on twinned crystals
I read your description of the crystals and Figure 4 of the following paper came to mind. Post-crystallization treatment in lower PEG eventually allowed them to tease the bundles apart. 1. MacRae, I. J. Doudna, J. A. An unusual case of pseudo-merohedral twinning in orthorhombic crystals of Dicer. urn:issn:0907-4449 63, 993–999 (2007). F On Feb 22, 2012, at 10:59 AM, Peter Hsu wrote: Forgot to mention, that this 2.5-3A diffracting crystal was the same one that I have been unable to index and suspect are twinned due to the presence of these other fused crystals in the same drop. Thanks for any input. - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] proper or improper ncs?
Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1:1.0.0. ROTA 2:0.1.0. ROTA 3:0.0.1. TRANS: 0.0.0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 TRANS:45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] offtopic : Signed binaries in the next OS X
It seems that Apple is building a higher walled garden for OS X in the form of signed binaries. They're not mandating every app come from the appstore but instead have a level that allows developers to 'sign' their binaries with their own developer ID (which of course costs $99USD/year). Or the user can go rogue and 'Ctrl-Click' install any application. http://www.apple.com/macosx/mountain-lion/security.html Personally I'll probably choose Mac App Store and identified developers. (I imagine this will be the default). - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] offtopic : Signed binaries in the next OS X
Hi Tim The problem is not developers ensuring their identities by signing their apps. It's that there's now a (small) barrier for the end user in installing unsigned apps. The implementation has yet to be seen, but will getting around this barrier simply be a pop up (press OK if you really trust this software, the implementation most people are familiar with but largely ineffective IMHO), or will the INSTALL file include OS X specific directives to circumvent the walled garden? (OS X users must CTRL-Click to install this application). You could sign your own software (for free) and then distribute your public key to the community, in case you want to do something similar. [FUD] OS X won't trust those keys, only the ones that come from apple [/FUD] F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] choice of wavelength
Acta Cryst. (1997). D53, 734-737[ doi:10.1107/S0907444997007233 ] The Ultimate Wavelength for Protein Crystallography? I. Polikarpov, A. Teplyakov and G. Oliva http://scripts.iucr.org/cgi-bin/paper?gr0657 may give some insights. To the OP, have you solved the structure? In some cases, seeing the packing at low resolution can give you ideas on how to change the construct to obtain higher diffracting crystals. F On Feb 15, 2012, at 4:21 PM, Jacob Keller wrote: Well, but there is more scattering with lower energy as well. The salient parameter should probably be scattering per damage. I remember reading some systematic studies a while back in which wavelength choice ended up being insignificant, but perhaps there is more info now, or perhaps I am remembering wrong? Jacob On Wed, Feb 15, 2012 at 5:14 PM, Bosch, Juergen jubo...@jhsph.edu wrote: No impact ? Longer wavelength more absorption more damage. But between the choices given no problem. Spread of spots might be better with 1.0 versus 0.9 but that depends on your cell and also how big your detector is. Given your current resolution none of the mentioned issues are deal breakers. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 18:08, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I would say the better practice would be to collect higher multiplicity/completeness, which should have a great impact on maps. Just watch out for radiation damage though. I think the wavelength will have no impact whatsoever. JPK On Wed, Feb 15, 2012 at 4:23 PM, Seungil Han shan06...@gmail.com wrote: All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Freezing crystal
I'm going with Jurgen on this one. http://img27.imageshack.us/img27/3232/pastedgraphic1.png Sad was the day when I mounted this puppy and it shot to 8-10A. Room temperature. And messing around with cryos didn't help either. Can't remember the size, but I think I had scooped it with a 0.8 mm loop. I should've mounted it on a ring and given it to my wife. And that's not chromatic artifact, the ligand was red. On a side note, I had a very small crystal embedded in a chunk of ice at the end of a 0.025 mm loop. Couldn't even see it on the very nice on-axis cameras at the ALS. I shot blindly into the ice.. .it diffracted to about 1.8A (and the ice wasn't bad at all) F On Feb 7, 2012, at 9:52 AM, Bosch, Juergen wrote: Hi Enrico, I was just looking at non-optimal cryo-conditions and the original posters starting point. Of course if you have a good cryo bigger is better for the reasons you write but if you have no clue how your crystals will perform then I'd rather go for small to be cautious and also have those around and not only the big ones which everybody mounts because they looks so nice. To be disappointed by big crystals is often not a surprise to me and if you have not tried small crystals from the same batch well then you missed 50% of your chances to solve s structure with the first light the crystals saw. Jürgen - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
On Feb 1, 2012, at 12:17 PM, Dianfan Li wrote: I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. 15 minutes is a long time. Scoop crystals during that time period. Do the cracked crystals diffract? Do you see the analogue? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] No diffraction
Ditto to Poul's advice. I've had many many many cases where crystals diffract poorly (or not at all) on home sources only to show excellent diffraction at a synchrotron. (Whether or not a home source is properly calibrated is probably the biggest issue, but that's for another discussion). On Jan 26, 2012, at 8:33 AM, Theresa H. Hsu wrote: Dear crystallographers I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and vapor diffusion method in 24 hours but no diffraction at home source. Dissolved crystals was confirmed to be the protein with mass spec. Any suggestions to improve diffraction would be welcome. Thanking you in advance. Theresa - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Merging data collected at two different wavelength
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Re: [ccp4bb] Merging data collected at two different wavelength
On Jan 18, 2012, at 10:20 AM, Tim Gruene wrote: Comments on the comments ;-): Ditto [...] By the way, I wouldn't use MAD to describe the mergeing of non-isomorphous datasets. I agree, neither would I. Just to be on the save side and avoid confusion by less experienced readers of the list: I used the term MAD because there are two data sets collected at two different wavelenghts, both of which should give rise to a measurable anomalous signal from the Co in the sample. Using the terms 'MAD' and 'SAD' have always been confusing to me when considering more complex phasing cases. What happens if you have intrinsic Zn's, collect a 3wvl experiment and then derivatize it with SeMet or a heavy atom? Or the MAD+native scenario (SHARP) ? Instead of using MAD/SAD nomenclature I favor explicitly stating whether dispersive/anomalous/isomorphous differences (and what heavy atoms for each ) were used in phasing. Aren't analyzing the differences (independent of source) the important bit anyway? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Choice of heavy atom derivatives
They show a nice diffraction, but appear to be perfectly twinned. In most cases, structures can be solved (and biological questions answered) in twinned cases. I wouldn't be discouraged. I have crystallised a SeMet derivative, but I have not been able to collect sufficiently good data, to get the phases. What is 'good' data to you? What are you considering as 'bad' data? I am a beginner in crystallography, so all your suggestions would be precious to me... You've come to the right place. Seek advice from people who have solved structures within the last 5 years using modern software. You'd be surprised how good the software for structure solution/refinement is these days. Thus, I was thinking of trying with some heavy atom soaks. This by chance is not a metalloprotein is it? No covalently bound heavy metals? (maybe you have done some biochemistry and found some heavy metal requirement for activity, purification, etc) which compounds and conditions would you advice as worth testing? If your native crystals diffract well on a home source, I would try soaking a fresh 500 mM solution of KI (iodide) for 5-10 seconds and collect the anomalous signal at home. If it kills your crystals, move to more toxic things... Thanks in advance, Federica Cheers, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] At what resolution is (individual,group,one per residue, two per residue) appropriate? was Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Pete brings out two concerns: [1] B-factor refinement being stable, [2] over-fitting at low resolutions. Here I'll suggest low resolution to be in the 3-4A range. I've seen the following question asked: At what resolution is (individual,group,one per residue, two per residue,overall) appropriate? One common response is try a number of different B-factor refinement protocols, use Rfree as a guide to determine which one is appropriate. I guess this is along the lines of B-factor refinement being stable. Seems reasonable to me. However is this approach sufficient justification to address over-fitting at low resolution? I'd welcome any thoughts on this ... F On Jan 6, 2012, at 11:00 AM, Pete Meyer wrote: B-factor refinement being stable is one thing; quieting my paranoia regarding over-fitting at low resolutions is another. Thanks for pointing this out to me - I'll have to check out the details of how phenix handles it, and give it a try. Pete - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] live streaming of ccp4 study weekend
The video works fine if you load http://extrplay.dl.ac.uk/CCP4/20120105-2/rnh.ram as a network stream into VLC (www.videolan.org) . Trying to play it with real player just didn't work. And VLC doesn't require your admin password to spew garbage all over your system. You don't get the jpg preview of the slides but at least the audio/video works on OS X 10.6.8. F On Jan 5, 2012, at 7:38 AM, David Mueller wrote: I can't get it to work on Firefox, Safari, or Chrome using a Mac with OS 10.6.8. David Mueller On 1/5/12 8:31 AM, Charles Allerston charles.allers...@sgc.ox.ac.uk wrote: How is everyone finding the stream? It was extremely flaky this morning here, cutting out every 15-20 seconds. The afternoon session doesn't seem to be faring any different at this end, sadly. Just wondering if it is a problem at our end or theirs. cheers charlie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of charles.ball...@stfc.ac.uk Sent: 04 January 2012 17:43 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] live streaming of ccp4 study weekend Dear All for all those not lucky enough to attend the CCP4 Study Weekend on Data collection and processing in Warwick there will be live streaming. This is at the link http://extrplay.dl.ac.uk/ kicking off with the what's new session at 9:00am GMT. The main event starts at 11:00am GMT. The full program is at http://www.cse.scitech.ac.uk/events/CCP4_2012/programme.html . All details are accessible from the ccp4 homepage (http://www.ccp4.ac.uk). Charles Ballard CCP4 - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] model building at 3.2 A
On the other hand, shooting a lower resolution crystal may get you the conformation of the disordered domain. Surprising at first thought, but was true in p97/VCP from the Brunger Lab. F On Dec 7, 2011, at 11:47 PM, atul kumar wrote: Dear all I have crytals which diffract up to 3.2 A at synchrotron, I am solving this by molecular replacement. I have built 70% of the model successfully,but the problem is it have a very poor density for some 30-40 residues at N terminal. I can't build anything in this region,this could be because of disordered structure or because of low resolution.what are the things which I can try to improve this? suggestion are requested! thanks regards' Atul Kumar
Re: [ccp4bb] Protein-DNA complex crystallization
Curious, how did you assess that your crystals only have DNA? F On Nov 21, 2011, at 8:59 AM, umar farook wrote: Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.coli ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] raw data deposition
On Oct 27, 2011, at 11:56 PM, James Stroud wrote: This is a poor criterion on which to base any conclusions or decisions. We can blame the lack of examples on unavailability of the data. Agreed. Reprocessing the data resulting in a a different biological result is my personal reason and motivates me to support raw data deposition. However, I'm not the one that needs convincing.. it's the other PI's/graduate students/postdocs (who may see MX as a simple tool to get an answer for some larger question), who need to see the value of depositing raw MX images. The point of my email is to elicit other people's reasons why raw data deposition is necessary. Right now, I'd love to get my hands on the raw images for a particular cryoEM data set, but they are not available--only the maps. But the maps assume one symmetry and I have a hypothesis that the true symmetry is different. I could test my hypothesis by reprocessing the data were it available. and thank you for providing yours . F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] IUCr committees, depositing images
This discussion of image deposition and archival has certainly been illuminating. While there have been clear directions on how the process would actually work, I am becoming increasingly curious on why it should be done (outside of threatening non publication, social acceptance, funding, etc). I think a huge challenge is to convince the users that filling out the metadata with as much detail as possible is a worthwhile endeavor. Specifically, are there documented cases where reinterpretation of an MX dataset (the raw images) has resulted in new biological insight into the system being modeled? If possible I'd like to compile a list (off list) for my own education (though I can certainly report my results to the BB if the interest is there). (Ok I have a selfish reason as well as I will be considering the reanalysis of some really old datasets that proved troublesome for a colleague and am looking for small hints of wisdom) Some that come to mind for me: Meyer et al. Structure of the 12-subunit RNA polymerase II refined with the aid of anomalous diffraction data. J Biol Chem (2009) vol. 284 (19) pp. 12933-9 Wang. Inclusion of weak high-resolution X-ray data for improvement of a group II intron structure. Acta Crystallogr D Biol Crystallogr (2010) vol. 66 (Pt 9) pp. 988-1000 F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] raw data deposition
Thanks for bringing this up front Ed. Specifically bringing your second point to the forefront. Do we need to do it? Or to rephrase it more directly .. WHY do we need to do it? Answering why we need to do it will really help with compliance. Lest we not forget we are asking the general crystallography community (which encompasses a large variety of interests in competition with the interest to archive the actual images) to go an additional step and provide detailed metadata (among other things). Of course you could force the community into compliance but I'm pretty sure we can motivate behavior without threats. So I ask again, are there literature examples where reevaluation of the crystallographic data has directly resulted in new biological insights into the system being modeled? On Oct 27, 2011, at 3:27 PM, Ed Pozharski wrote: 1. How to do it. That is what the other thread is dealing with and my overall feeling is that difficulties have been largely exaggerated early on. You are right that concrete steps can be taken. 2. Do we need to do it. To me, it's no-brainer, but some responses seem to suggest not everyone is really on board. Again, I am sure this has to be done, but consensus in this area is equally important. - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] solvent mask from one crystal form applied to the other second ...
Hi Pete Thanks for the reply. As for the transformation, I was going to use the refined mask operator (either from the end of a DM run that produces a better map for features I know have to be there, or from a correlation of the averaging mask from MAVE). The interpolation is going to be a little more difficult. The 'good' solvent mask output after a dmmulti run has different gridding, extent, and cell than the mask output by the other crystal forms. However, the documentation for dmmulti says that SOLin can be on any grid or axis ordering.. not quite sure about the extent. Furthermore, it seems that Poul Nissen was able to put in a generic mask for all his crystal forms (http://journals.iucr.org/d/issues/2010/03/00/kw5018/index.html) without regard for interpolation (unit cell, extent, or gridding) nor transformation. So I'm not exactly sure how I'm getting an 'inconsistent cell info'. Maybe it's because I'm getting my masks from dm instead of generating it from a my averaging mask (which came from a pdb).. hmmm... Anyone know of a wiki page that talks about maps/masks and a description of their properties (gridding, extent, basically everything discussed in the output of mapinfo?) A wiki with examples (images) of different settings for these items would be especially useful. F On Oct 24, 2011, at 9:40 AM, Pete Meyer wrote: Hi, It's definitely possible, but the devil is in the details. It's a two-step process: transform the coordinates of the mask so it's in the right spot in the new cell, and interpolate the mask into the appropriate grid for the new crystal form. The last time I did this I was using O/PHASES masks (so I'm probably off on the details for CCP4 stuff): maprot should be able to handle the transformation and interpolation, but you may have to use a mask - map - maprot - mask pathway. This isn't ideal due to differences in mask interpolation vs map interpolation but could be a good starting point. Determining the transformation may be tricker - I'd try placing pseudo-atoms at similar features in both maps and using lsqkab to get the transformation matrix; but this approach would probably need tuning to get a good alignment. Good luck, Pete Francis E Reyes wrote: Hi all I'm using dmmulti and one of my crystal forms seems to have a better solvent mask (after outputting it with solout) than the other. (Probably due to better phased reflections at low res for the first crystal form). Is it possible to take the better solvent mask and use it as input for the solvent mask for the other? How's this done? (a direct input of the better xtal1 mask to xtal 2 gives 'inconsistent cell info'). Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] how to input O-style rotation matricies in dmmulti?
All I have an O-style operator but when I put in the following under dmmulti, dm refuses to continue to read the rest of my instructions...? AVER - REFI omat 1.0 0.0 0.0 0.0 0.62 -0.008727 0.0 0.008727 0.62 -0.215399 -0.909085 -0.017797 XTAL 3 (it never reads XTAL 3). Is there some formatting error I'm not seeing? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Ice rings...
All, So I have two intense ice rings where there appear to be lattice spots in between them. I understand that any reflections that lie directly on the ice ring are useless, however, how do software programs (HKL2000, d*Trek, mosflm, XDS) deal with these intermediate spots? It would seem to me that employing a 'resolution cut off' just before the ice ring (on the low resolution side) would be improper, as there are spots on the high resolution side of the ice. (see enclosed .tiff) In fact, how do these programs deal with spots lying on ice rings? Are they rejected by some algorithm by those programs during integration, or is it up to the scaling/merging (by SCALA for example) step to deal with them? Thanks! F inline: PastedGraphic-1.tiff - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] off topic: Adobe demos deblur of photos...
http://hoowstuffworks.blogspot.com/2011/10/adobe-demos-amazing-unblur-feature.html Though I can't really see the image myself... the gasp of the audience is telling With respect to existing density modification programs, I wonder if such technology (whatever it is) can ever clear up my messy density maps. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] XDS suggestions: Frame.cbf shows white circles in the beamstop...
I already started with an ellipse... should I extend with a rectangle down the shaft of the beamstop?(this is FRAME.cbf after integration) inline: PastedGraphic-2.tiff Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Linux vs MacOS for crystallographic software
Bill Thanks for focusing the thread to the original poster: If you're going to go OSX I would wary away from the iMac. The all-in-one desktop solution in small form factor has its downfalls, particularly when the mechanical disk (undoubtedly) fails. I have an iMac from 2007 and the hard drive needs to be replaced. The rest of the computer is fine and will probably work for the next 3-5 years for light desktop work (at least for the wife and kiddos). However, I expect the disassembly just to replace this component to be a nightmare. F On Sep 30, 2011, at 9:44 AM, William Scott wrote: she asked were a few questions about a specific computer (HP Z210 8 GB with a low end Quadro Nvidia 400 512 MB) running any Linux, and a specific computer (IMAC 4 GB 2.5 GHz with AMD Radeon HD 6750M 512 MB) - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Linux vs MacOS for crystallographic software
On Sep 30, 2011, at 12:07 PM, Adrian Goldman wrote: I would disagree about the disk issue. That's not the failure mode we have seen in the iMacs. Fwiw. Anyway, if it were to fail you could just attach an external disk and continue merrily along - macs will boot from external FireWire (and I assume thunderbolt?) disks. Yes, but I treasure my desktop and USB/Firewire port space ;) My point is that replacing anything (video card, logic board, display card, drive) within an iMac is difficult. If the OP (like me) plans on keeping your computer for more than 5-7 years, then he/she might want to get something where replacing the hardware is easy. I still have several fully functional dual processor PowerPC G5 from 2004 that work wonderfully for general desktop use. And it will still run most (CCP4/Phenix/Coot) crystallographic software. We are putting money where my mouth is. Our last five purchases have been i7 iMacs. It seems like quite a nice amount of oomph for the money. Core i7? Then your macs are still quite young (i7's were introduced in 2010). I'm talking a core 2 duo mac from 2007 (the iSight G5). Time will tell if the drives in 2010 were any better than then drives from 2007. I doubt it though. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] neutron diffraction 'difference' map?
Hi all Suppose you have a high resolution xtal structure (from usual x-ray diffraction) and you wanted to verify the location of a ligand. You can purchase a heavy atom isotope version of the ligand. [1] Is it possible to do a neutron diffraction difference map (where you simply calculated Fobs(heavy) - Fobs(light) to verify the location of the ligand? [1b] Suppose you couldn't measure the diffraction of the light version of the ligand. Can the x-ray data be combined with the neutron diffraction of the heavy atom isotope to unambiguously assign the ligand site? [2] What would be the minimum resolution required from the neutron diffraction? (This is particularly important as you may be unable to grow high diffracting crystals or large crystals) Note that you don't necessarily want to solve the structure of the structure from neutron diffraction from scratch, but rather you want to use it as a tool to verify the location of a ligand binding site. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] refmac and DNA (and now RNA)
Is ' U ' now the standard vs ' U' ? I'm used to right justified letters for RNA residues in the residue field. This is with a recent refinement with refmac 5.6.0117 . And of course this switch in naming convention breaks compatibility with molprobity (which requires right justified letters in the residue field) F On Sep 8, 2011, at 7:35 AM, Ed Pozharski wrote: After switching (finally) to 6.2.0 and therefore to Refmac 5.6.0117 I have found a problem working with DNA that I have not seen with 6.1.13/5.5.0109. Namely, - if I use the pdb file produced by Coot (0.7.pre-1.3470) that seems to output DNA as Ad/Td/Gd/Cd no matter what the input names were, refmac fails with the warning that it found a new monomer. It appears that it stumbles upon the very first thymidine, but in a strange twist it reports the problematic residue having the name DY! - if I use the pdb file previously produced by refmac, which has the A/T/G/C as residue names, it fails too but now complains about the new monomer named T. - the workaround I found is to rename all the thymidines to DT. It is a bit annoying since coot keeps renaming them (well, not refmac/ccp4 problem per se) and I have to rename back (easily scripted task, of course). What is peculiar is that Ad/Gd/Cd don't need to be renamed (does this have anything to do with thymidine being the only one that changes residue name in RNA?). Has anyone else seen this or it's something specific to my setup? Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] refmac and DNA (and now RNA)
Hi Garib Thanks for the quick reply! Refmac however is writing my pdb's as with the residue letter in the centered position. Is the newest pdb requiring centered residue letters for RNA? F On Sep 12, 2011, at 1:05 PM, Garib N Murshudov wrote: Refmac will read right or left justified residue names, however pdb may use only one of them. - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] How to handle b-factors at low resolution and very incomplete model?
Hi all What's a way to determining a B-factor to set for all residues of a model at low resolution (4A)? How valid is the wilson B at this resolution? (better than nothing?) I'm in the process of fitting whole domains into some rasty experimental density maps and was thinking of doing a rigid body or very restrained ( secondary structure | reference structure | etc ) refinement of the coordinates for helping the fit. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Modeling ligands in binding pockets when the density is weak.
Seems to be a quiet day on the BB, so I propose this question: Suppose you have a ligand in the binding pocket and some mediocre data (3 A or so), the 'core' of the ligand is well defined in 2Fo-Fc map using the model phases of your protein, however there are 'chains/tails' of the ligand which are not. Composite omit or simulated annealing omit maps do not produce density for these 'chains' The question here is how the chains/tails should be modeled (if at all). [1] Model in the core, but remove the atoms for the chains (and conclude the diffraction data do not support interactions with the protein and subsequent experiments are needed (higher resolution data, biochemical data, etc)). or [2] Model in the chains/tails noting that potential hydrogen bond donors/acceptors on the protein are within hydrogen bonding distance to the chains/tails. You do this and subsequent refinement still does not produce the expected density for the chains. or [3] Your solution here. If this situation has been discussed before, please let me know . F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] COOT library
Welcome to the quirks of RNA crystallography. It is a small community in a sea of protein crystallographers so expect to get used to work arounds to get things to work. Your answers are at the coot mailing list archives (we recently had a discussion regarding coot 0.6.2 ver 3562) . https://www.jiscmail.ac.uk/cgi-bin/webadmin?A1=ind1108L=COOT F On Aug 17, 2011, at 10:10 AM, Eric Karg wrote: Thanks for the suggestions. I could solve the problem by using the setenv command to the coot directory,but I still cannot use the Real Space Refine Zone in Coot because the names are not compatible. I'm trying to build an RNA molecule by the way. Any suggestions? Eric - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Good performing low resolution iterative model building programs?
Hi ccp4bb'ers, Of the automatic model builders out there (autobuild, arp/warp, buccaneer, insert your own here), are there any opinions/personal experience on which of these perform well in low resolution cases ( worse than say 3.5-3.8 ) ? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] anomalous scatterer
On Aug 9, 2011, at 10:37 AM, Huiming Li wrote: Hi All, I am working on a Tb binding protein on which I collected anomalous data at Tb edge of 1.648 A. Each protein is designed to bind one Tb. There are two copies of the protein in an ASU. I have two questions. First, I am only able to see one copy of the protein with Tb bound, and no density on the other copy. Isn't this a bit surprising? I'm assuming that when you say 'density' you're referring to the density around the Tb in both proteins. Radiation damage? One bound Tb and not the other? Second, there is one additional peak on each monomer at the site where Fe is known to bind, and Fe has an edge of 1.739A. At 1.648A, f' and f'' of Fe is only about 1/5 of Tb. Is it possible Fe also shows some anomalous signal at Tb edge? Depending on the corectness of the (experimental or model) phases, I've seen Co at the Ir peak and Ir at the Co peak. Use both to cross validate your model (when you get to it) and calculate experimental phases using both (dual SAD or 2wl MAD for each heavy atom). So no it's not surprising. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Multi crystal averaging : data on same scale before averaging?
Hi all Walking through multi xtal averaging with RAVE. I finally got a good mask and optimized NCS for my xtal forms. However, in the CRAVE manual I see this - the reflections in the input MTZ files *MUST* have been put on the same temperature factor scale prior to cross-crystal averaging (see the DATAMAN manual on how to do this) !!! Scale the separate datasets together? Wouldn't this just be a mess since the crystal should be non isomorphous to each other? Or does this say that within each dataset, all the data should be on the same scale (which would be if I used scala to scale) ? Am I interpreting this correctly? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Way off topic.. less than 96 well culture plates in black?
Hi all Sorry for the off topic post, but given the breadth of experience on this bb, I would like to ask. I need to measure fluorescence from bacterial cell cultures in a 12/24/48 cell culture plate (the number of wells is unimportant). As this is a fluorescence measurement, I require them to be black on the walls. I'm looking for a supplier / catalog number of such plates. To save bb spam, I'll take replies privately. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
Never had problems with evaporation (and this is in the relatively dry climate of Denver, CO, especially in the winter when the relative humidity is in the low 20%). Using the Thermo Scientific Nanodrop 2000c. We use it also as a prerequisite for ITC, which can be very sensitive to proper concentrations. F On Jun 16, 2011, at 1:15 PM, Arnon Lavie wrote: Dear fellow crystallographers - a question about spectrophotometers for protein concentration determination. We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein conc. determination. We have been considering buying a Nanodrop machine (small volume, no dilution needed, fast, easy). However, while testing our samples using a colleague's machine, we have gotten readings up to 100% different to our Bradford assay (all fully purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how reliable are the measurements (your thoughts?). So QUESTION 1: What are people's experience regarding the correlation between Nanodrop and Bradford? While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter Pearl. So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose? Thank you for helping us to advance to the next millennium, even if it is nearly a dozen years late. Arnon -- *** Arnon Lavie, Professor Dept. of Biochemistry and Molecular Genetics University of Illinois at Chicago 900 S. Ashland Ave. Molecular Biology Research Building, Room 1108 (M/C 669) Chicago, IL 60607 U.S.A. Tel:(312) 355-5029 Fax:(312) 355-4535 E-mail: la...@uic.edu http://www.uic.edu/labs/lavie/ ***
[ccp4bb] confused about ncs in this xtal and local correlation maps with maprot.
Hi all I was just fiddling around with ncs and maps, so I tried to use maprot a 2fofc map (refined model) of a crystal that has two molecules related by the NCS: (given by LSQKAB over the CA's) CROWTHER (Euler) ALPHA BETA GAMMA 95.8381478.49123 145.66571 TRANSLATION VECTOR IN AS 95.52723 -43.90490-3.70434 Loading the resultant PDB from lsqkab, overlaps the other molecule perfectly. However, when I try to load the local correlation map, it's quite poor (borderline nonsensical). (And this is done for a number of sphere's from 4-10A, the resolution of the data is about 3.1A). The unit cell is: 142.0500 142.0500 137.9200 90. 90. 120. P6422. Any ideas appreciated, Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] Where is multillog for dmmulti?
Can someone return their full path of the location of multilog (used for browsing dmmulti output)? I can't find it in my ccp4-6.1.13 installed from Mr Scott's distrib. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] early (incomplete model) refinement if you had the location of a bound (highly occupied) anomalous scatterer
Hi all, could you use its position in real space as a target for (to make it easy, rigid body) refinement? Real space meaning the electron density around the scatterer in an anomalous LLG map. Some other restraints: Say it's a metal cofactor and you know that it needs to be in a specific position with respect to your protein. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] molecular replacement solution
Is there NCS? (How's the self rotation look? How about the native patterson?) Does your search model have flexible loops? You may have the right solution, but the flexible loops may be in a different conformation and cause clashes. Trim the search model to the conserved / structured domains. Do you have *any* anomalous signal? (whether you collected Sulfur-SAD, you have a metalloprotein, etc). These can be useful in verifying the correctness of the MR solution. I use only one chain of the monomer model Why? F On Mar 15, 2011, at 12:41 AM, Careina Edgooms wrote: Dear CCP4 members I have a confusion with molecular replacement. I wish to solve a monomeric protein in P21 where there are 2 monomers in asymmetric unit. I have a search model that also has 2 monomers in asymmetric unit. First I try using Phaser. I use only one chain of the monomer model and search for 2 copies. This gives a good solution that refines with low R-factor but it shows many clashes. In other words, the 2 monomers are too close together and there are clashes where they meet. Then I try using Phaser and both chains of the model and search for 1 copy. This does not give a solution and says asymmetric unit is too full. Then I try using MolRep with the 2 chain search model. It gives solution that looks better because you can see that the 2 monomers do not physically clash but it refines very badly giving R factors of about 50% So I am confused. What am I doing wrong? Please help me with suggestions Many thanks Careina - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] molrep NOSG=-1 (space group checking)
Hi all I'm checking all space groups under P222 for data that contains a pseudotranslation. The data integrates in P222 but a 26% PST peak (0.500, 0.000, 0.23) makes this look like a C2221 cell (see previous CCP4bb post subject: Let's talk pseudotranslational symmetry (or maybe it's bad data). I was able to get a solution (2 molecules per ASU related by PST) in P 21 21 21, and able to build into additional density and refine to R/Rfree of 0.274/0.317 for 3.5A data. There still a few uninterpretable blobs (a linker region of about 6 residues) left. I'm now trying to do the MR of the refined model in all combinations of P222 via MOLREP (NOSG=-1). Now I'm trying to interpret the output of molrep. --- Space Group Checking. --- I,Nsg,Scor,Cntr:1 16 P 2 2 2 0.375 5.521 I,Nsg,Scor,Cntr:2 17 P 2 2 21 0.415 12.143 I,Nsg,Scor,Cntr:3 1017 P 21 2 2 0.337 1.722 I,Nsg,Scor,Cntr:4 2017 P 2 21 2 0.327 14.237 I,Nsg,Scor,Cntr:5 18 P 21 21 20.419 5.169 I,Nsg,Scor,Cntr:6 2018 P 21 2 210.414 10.808 I,Nsg,Scor,Cntr:7 3018 P 2 21 210.463 12.357 I,Nsg,Scor,Cntr:8 19 P 21 21 21 0.621 18.538 Time:11h 22m 8s Elapsed: 0h 11m 30s MOLREP(ccp4): Failure [1] Is there somewhere in the man page or documentation that explains how 'Scor' is computed? [2] Based on the results above, it seems that P 21 21 21 is the correct s.g.? [3] Why the failure? Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] inverse beam integration/scaling protocol
Hi all Is there a walkthru/tutorial for processing inverse beam images with imosflm/scala? Googling a few things didn't get me anywhere. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] CCP4 SW Web Broadcast
Video is fine.. Anyone getting audio (or is it my realplayer setup? ) ... F On Jan 6, 2011, at 10:04 AM, ronan.kee...@stfc.ac.uk wrote: Dear all, The web stream is now working and can be accessed from: http://extrplay.dl.ac.uk/ There will be full coverage tomorrow (Friday). Apologies again for the delay. Best wishes, Ronan -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ronan.kee...@stfc.ac.uk Sent: 06 January 2011 08:50 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] CCP4 SW Web Broadcast Dear All, For those of you hoping to view the CCP4 Study Weekend online, we're experiencing some technical difficulties with the webcast equipment. Unfortunately the live stream won't be available for the first part of the meeting. We're hoping to fix it as soon as possible and we'll send an update as soon as it's available. Our apologies for the inconvenience. Best wishes, Ronan Ronan Keegan CCP4 Group - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Mg2+ or water
If it's more than likely Mg and it's easy to grow crystals, try soaking/cogrowing BaCl2/BaOAc. At least in the RNA world, Mg sites can easily be displaced by Ba. The latter of course has anomalous signal on the home sources. Place your divalents using the anomalous diff map. Have no clue what this .odp file ext is so I can't view the png. F On Dec 20, 2010, at 2:16 PM, jlliu liu wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H- bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL test.png.odp - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] off topic: advice for crystallography workstation/server
Ronnie Just a few comments.. There are no 'true' virtual sessions for OS X Server. (Well there is, but it's developed by a separate company and they charge per seat == expensive). What I define as a 'virtual session' is that you get a login window and your own desktop all running over VNC. (and you can do this for multiple users). The Apple Remote Desktop software only shares one screen/one user. Just as the other commenter said, multiple users on a crystallographic workstation is moot if you need 3d or even graphics intensive visualization (as each computer would benefit from having coot/pymol run locally). The number of programs taking advantage of multiple processors is growing, but far from mainstream (support, software usability, benefits etc). Much of crystallography remains to be a 'serial' rather than a 'parallel' experience. That being said, each your money may be better spent giving each user a Mac Mini (the price for 3-4 Minis == 1 Lowest end Mac Pro) .. and if you truly want multiple user management get a Mini Server that does manages user accounts/policies with local home directories. You could even keep the software on the main server and map it to the clients so you have version control over the xtal packages. F On Dec 15, 2010, at 9:26 AM, Ronnie Berntsson wrote: Dear all, We are currently considering buying a computer which can be used by multiple people, via our existing network, as a workstation for crystallography purposes. My thoughts are currently going towards a 8-core Apple Pro (or 12-core) with a lot of RAM, with OS X Server, which in theory should be able to handle multiple (up to 4) users simultaneously running crystallography software. The idea would be to have the users access this computer using their own laptops (starting their own virtual sessions?) connected to the same network. Does this sound like a viable strategy, or should it be setup in a different way? In that case how? Would it need advanced setup and maintenance, or would it be possible to jsut set up a number of user accounts in OS X Server, and let it run? I'm reasonably computer savvy, but haven't really done something like this before, so I would very much appreciate your advice or personal experiences regarding this matter. I know that I could probably get a cheaper computer if I went for a pc with linux, but I have more experience with OS X, and would therefore want to stay with it. Thank you in advance, Ronnie Berntsson -- Ronnie Berntsson, PhD PostDoctoral Fellow Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute Zernike Institute for Advanced Materials University of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] ncs operator confusion...
Hi all I'm trying to figure out rotation matricies for ncs averaging in DM. Except the conventions have got me confused. The following input script for pdbset correctly maps monomer A to monomer B. rotate - invert - -0.4654 -0.0974 -0.8797 - 0.8789 -0.1689 -0.4462 - -0.1051 -0.9808 0.1642 shift - invert - -108.0451 47.655 -17.5654 end Can someone supply me with the correct matrix to input into DM (and what convention it is?) Thanks F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] ncs operator confusion...
Oops It pays to view the pdbset log as it gives the right matrix. F On Dec 1, 2010, at 10:41 AM, Francis E Reyes wrote: Hi all I'm trying to figure out rotation matricies for ncs averaging in DM. Except the conventions have got me confused. The following input script for pdbset correctly maps monomer A to monomer B. rotate - invert - -0.4654 -0.0974 -0.8797 - 0.8789 -0.1689 -0.4462 - -0.1051 -0.9808 0.1642 shift - invert - -108.0451 47.655 -17.5654 end Can someone supply me with the correct matrix to input into DM (and what convention it is?) Thanks F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] limit for number of files for CAD...
CCP4'ers, Are 9 datasets the maximum for an mtz file? or a single run of cad? The manual (http://www.ccp4.ac.uk/html/cad.html) seems to suggest that 9 is the limit per cad run but not for a given mtz. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Crystal lattice builder (educational)
Hi all Are there any online/offline 3D crystal lattice builders to explore symmetry relationships in the unit cell given say a space group and unit cell parameters? I need one for an educational opportunity. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
Hi all I'm reading a recent review by Elspeth Garman regarding radiation damage (Acta Cryst D) and in this she mentions two ideas regarding metal centers: [1] They (The metal complexes themselves) are quickly reduced [2] Their absorption causes localized heating in the crystal. (This causes the temp to increase above freezing thus allowing OH radicals to diffuse). How is it that people can phase off of TaBr considering [1]? Presumably as TaBr is absorbing x-ray's whatever anomalous signal that was present at the beginning of the collection is either absent/ significantly altered by the time data collection is complete? Regarding [2], is TaBr usually backsoaked (into cryoprotectant not containing TaBr) to reduce it's concentration in the crystal to highly occupied sites? It would seem to me that having a crystal soaked with TaBr would cause more heating than one backsoaked. Thanks F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] difficult P1 crystal
What does this mean? You index what looks to be tetragonal, do your collection, and then it integrates/scales only in P1? F On Sep 30, 2010, at 5:54 AM, Mario Milani wrote: It initially looks like tetragonal (I4, a=141, b=141, c=208) and then results triclinic (P1, a=141, b=141 c=144, alpha=119, beta=119, gamma=90), - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
Hi all I'm interested in the scenario where crystals were screened at home and gave lousy (say 8-10A) but when illuminated with synchrotron radiation gave reasonable diffraction ( 3A) ? Why the discrepancy? Thanks F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] offtopic: effect of compound impurities on ITC?
Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] offtopic: adjustable vertical sequencing gels.
All, Sorry for the off topic/product solicitation, but someone may be able to help. Looking for an adjustable sequencing gel that can replace the ADJ3 from Thermo Scientific. (http://www.owlsci.com/verticals/vertical.aspx?id=ADJ3 ) . Unfortunately, it's been discontinued. Needs to be able to accommodate 35 cm (wide) by 26-30cm (height) plates. Please reply privately. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Stuck refinement
Sorry a late comer to this thread but the OP mentioned tweaking the error model in HKL2000. I have heard this before.What's the validity in this? Does it actually help or does it only help the integration numbers but you'll pay for it during refinement? FR On Jun 23, 2010, at 8:25 AM, Zhou, Tongqing (NIH/VRC) [E] tz...@mail.nih.gov wrote: Hi All, The problem has also been solved with a new 2.0A dataset collected over the last weekend. Same space group and dimensions, much less radiation damage. This time I used APS SER-CAT's weaker BM beamline. Thanks, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ** -Original Message- From: Zhou, Tongqing (NIH/VRC) [E] Sent: Tuesday, June 15, 2010 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stuck refinement Hi, Everyone, Thank you all very much for the nice suggestions. I am trying to reply within this email. I agree that the problem may be rooted from the crystal itself, we noticed during data collection that a wedge of the rotation was very mosaic, HKL2000 was able to pick up the right spots, but the scaling gives high chi^2, and when I used the rejection files, HKL2000 complained more than 5 rejections. Colleagues suggested tweaking the error model, the complaint of more than 5 rejections' went away and rejection dropped to below 300 spots. The new error model reduced the chi^2 as well as the I/sigI in the low resolution shells. I run the P222 data set with Xtriage, the report says no twining, but the symmetry was too low. However, HKL2000 won't even pick up higher symmetry groups during indexing. I also rescaled the data omitting the bad wedge, xtriage gives normal report. Refinement was done with combination of simulated annealing, TLS, ADP, individual sites in Phenix. The molecular replacement was done with CDR-loop-trimmed antibody Fab and antigen structures. The map quality was good and I was able to rebuild the new loops without any problem. I will have beam time later this week, I think it will be better to put a better crystal on. Best regards, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ** -Original Message- From: Eleanor Dodson [mailto:c...@ysbl.york.ac.uk] Sent: Tuesday, June 15, 2010 4:46 AM To: Zhou, Tongqing (NIH/VRC) [E] Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stuck refinement When this happens, I firstly suspect that the spacegroup may be wrong. We had a case where the symmetry was pseudo I4212 but was really I222 (or was it really I212121) Anyway most of the structure obeyed the I41212 symmetry but there was a tail which did not..) Feed the unmerged reflections into pointless and see what it suggests Eleanor Zhou, Tongqing (NIH/VRC) [E] wrote: Hi Everyone, I have some problem in refining a structure. The data goes to 2.4A (with some 30% completeness at 2.15A), the structure was solved by MR with Phaser, refinement was done with Phenix, but the r and r- free are now staying at 26% and 32%, even with all possible waters and missing fragments added. Data was collected at APS at cryo condition. One thing I noticed during HKL2000 data processing was that the chi^2 were way too high at lower resolutions shells, I
[ccp4bb] Charge flipping.
Hi all I've been playing around with charge flipping for macromolecular substructure determination with pretty promising results. I'm particularly attracted to the fact that it solves structures in P1, with no space group assumptions and curious how it would handle some of the pseudosymmetry cases I've come into in my time. I'd like to know if anyone's had experience with this method, and open up the discussion with the following questions: As the algorithm starts with completely random phases and charge flips the map in P1, what is the importance of measuring (good or any) anomalous signal at all (for the sole purpose of finding the heavy atoms)? At first pass it would seem that just as long as you have an incorporated heavy atom and the density of that region is greater than delta, that this alone would be sufficient for locating the position of the heavy atom. In other words just as long as your heavy atom is sufficiently higher in contrast than your protein/rna it would be a good enough criteria. In the above regime, would the importance of measuring anomalous data be more important for substructure refinement (via phaser, mlphare, sharp, solve/resolve)? Now to a more specific question for those who've had experience (or maybe the authors are subscribed here): Orthorhombic C2221 using SUPERFLIP heavy atoms are found with great peakiness (before noise suppression: peakiness = 5, after noise suppression peakiness 25, good separation of heavy atom peaks from noise peaks in resulting pdb). Yet the space group check via the sym operators is rather poor (overall agreement close to 100). My interpretation is that the heavy atoms are found, but the space group is wrong? Thanks! F - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] odd request: add phase error linearly with resolution
Hi all I'd like to add a phase error to my PHIB's and FOM's (experimental phases) that increases linearly with higher resolution.. it's akin to taking good phases and making them bad. Any approaches on how this can be done? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] self rotation education
Hi all I have a solved structure that crystallizes as a trimer to a reasonable R/Rfree, but I'm trying to rationalize the peaks in my self rotation. The space group is P212121, calculating my self rotations from 50-3A, integration radius of 22 (the radius of my molecule is about 44). I can see the three fold NCS from my structure on the 120 slice, but I'm trying to rationalize apparent two folds in my kappa=180. A picture of both slices is enclosed. The non crystallographic peaks for kappa=180, P222 begin to appear at kappa=150 and are strongest on the 180 slice. My molecule looks close to a bagel (44A wide and 28A tall). The three fold NCS is down the axis of looking down on the bagel hole. I'm trying to find the two fold. I imagine it could be slicing the bagel in half (like to eat it for yourself) or slicing it vertically (like to share amongst kids) but I'm not exactly sure what's the best way to visualize this. Is there something easier than correlation maps with getax (since I have the rotation (polarrfn) and translation?). If you have an eye for spotting symmetry, Ill send the pdb in confidence. Thanks! FR inline: 120.jpeginline: 180.jpeg - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] density in LLG maps for heavy atoms sit on xtal axes?
Is this a cause for concern? FOM's are over 0.5 and Phasing Power is over 2.0. Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] low resolution phasing protocols
Hi all I'm in search of literature *detailing* low resolution [4-5A] phasing protocols. Heck I'll take detailed thesis chapters (since they tend to be more detailed than pubs anyway). If you ribosome people are on this board I'd love to hear from you. [sarcasm]Searching for 'grasping at straws', 'last ditch efforts', 'making something out of nothing' failed to yield anything productive[/sarcasm]. By detailed I'm hoping for discussions on how the authors knew progress was being made, software and scripts used, etc. Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] how to supply the following twin operator to detwin?
Hi All I'm having trouble with the following ... title [No title given] operator -1/2*h+1/2*k-l, 1/2*h-1/2*k-l, -1/2*h-1/2*k labin I(+)=I_Unspecified(+) SIGI(+)=SIGI_Unspecified(+) I(-)=I_Unspecified(-) SIGI(-)=SIGI_Unspecified(-) end I keep getting symmetry operator error. Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Omit maps in cases of NCS?
How does one calculate a simulated annealing omit map in cases of non crystallographic symmetry? The omit map is being used to identify the presence and configuration of a ligand and surrounding residues. The omit region therefore will contain the ligand and residues within hydrogen bonding contact. The residues obey NCS. The refinement will not include NCS ( refinement at 2.0 A with ncs leads to worse r/rfree statistics). with that said should the omit region include all NCS related molecules or just one? If the former, does one average the sa omit mAps to present in a figure? Thanks, FR
[ccp4bb] Experience with experimental phasing in P21 with c cos Beta = - a/2 ?
Or know of any references besides Declercq and Evrard, 2001 ? I'd love to hear from you. Please PM me. Cheers FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Reindexing P21.. the process?
+1 phenix FTW FR On Jan 13, 2010, at 3:28 PM, Peter Zwart phzw...@lbl.gov wrote: try using phenix.reflection_file_converter data.sca --change_of_basis=h,-k,-h-l --sca=reindex.sca or something like that HTH Peter 2010/1/13 Francis E Reyes francis.re...@colorado.edu: Hi all I have data integrated and scaled to P21 via denzo/scalepack. I'm concerned about the workflow to obtain the alternate indexing arrangement (h,k,l) - (h,-k,-h-l). I was thinking .sca ( not specifying NO MERGE) - .mtz - reindex but the documentation for reindex says all my DANO columns are 'negated' ( I do not specify NOREDUCE). So I must run truncate on the reindexed intensities to recalculate F(+/-) and DANO/SIGDANO) ? Thanks FR -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] How to reduce R-factor and Free-R
What is the space group? Is there unmodeled density? Are all residues in your construct built? What is the refinement protocol? FR On Dec 18, 2009, at 6:47 AM, james09 pruza james09x...@gmail.com wrote: Dear All, I am trying to solve a 2.55 A resolution data set. The R-factor is around 24% while Free-R is 30%. The Residues in most favourable region is 95% while additionally allowed region is 5%. What are the ways to reduce the R-factor and Free-R? Around 100 water molecules are placed. Thanks in advance for the help. J...
[ccp4bb] optimizing ncs masks in DM + interpreting output
Hi all I have a rotation + translation component for the 2-fold given to me by GETAX using low resolution low quality experimental maps. I understand that these are input into DM for NCS averaging. What are signs of progress as well as how does one usually optimize the mask? When doing automask is there a certain value of the masked region or correlation that is considered acceptable? I'm getting some refined NCS matricies that have a decent correlation (0.275-0.344). I'd like to see if it's still refining an NCS operator that is consistent with the self rotation maps. The self rotation maps for my two fold ncs show a euler coordinate (alpha beta gamma) of 0, 90, 180 which is 48.4% of the crystallographic peaks. The refined NCS operator from DM is 15.374 73.253 164.235 showing some deviations from pollarrfn. Is this an indicator of epic failure? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] offtopic: screening compounds libraries for binding via crystallography
Hi All I'm interested in screening small molecule libraries against my xtal for potential binders. There are many papers that cover this (http://www.nature.com/nbt/journal/v18/n10/abs/nbt1000_1105.html , for example). If you have had experience in this field, please email me privately as I would first like to see how feasible it is to perform these experiments (especially on an academic budget). Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] fix or scale bfactors during processing?
Hi all I have pretty low res data (4-4.5A) and was wondering what's the suggestion for allowing the bfactors to vary or keep them fixed during scaling. If I fix them, xtriage reports wonderful anomalous signal whereas if they are varied, the anomalous signal is gone. I have natives and suspected derivatives so MIR is possible. Rmeas's are pretty similar independent of whether I keep them fixed or allow them to vary. Is there a more sensitive statistic (to whether or not to scale or vary bfactors) that I'm missing? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Using own orientation matrix with imosflm.
Hi All I have a predetermined matrix from labelit, how can I use it with imosflm? I add the matrix file under 'Images', but I cannot integrate. Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] multiple datasets in Xds
Hi all Can XDS take in additional images that have a different template name and reside in a different directory? The reason is that I have high res images and then low res images in different directories that I want XDS to consider. Thanks! FR
[ccp4bb] Interpretation of wilson statistics and pseudosymmetry in R32.
Hi all I'm working on a structure of an RNA. Phases were obtained via SAD and the structure was refined to 21/26 % on R and R-free with a resolution of 2.8A, single molecule in the ASU. 7 residues were missing in a disordered loop out of a total of 54 residues in the asu. The cell was R32, dimensions 105.3598 105.3598 107.7804 90. 90. 120.. Xtriage showed decent statistics for this dataset: Wilson ratio and moments Acentric reflections I^2/I^2:2.060 (untwinned: 2.000; perfect twin 1.500) F^2/F^2:0.779 (untwinned: 0.785; perfect twin 0.885) |E^2 - 1|:0.759 (untwinned: 0.736; perfect twin 0.541) Centric reflections I^2/I^2:3.131 (untwinned: 3.000; perfect twin 2.000) F^2/F^2:0.653 (untwinned: 0.637; perfect twin 0.785) |E^2 - 1|:0.937 (untwinned: 0.968; perfect twin 0.736) We were able to obtain crystals that shot to about 2.0A, again in R32, with the unit cell dimensions 103.5 103.5 108.888 90 90 120. Refinement of the low resolution structure into this high resolution dataset proves to be problematic. The last 7 residues cannot be built without building into a symmetry related molecule yet there is uninterpretable positive density in the 2Fo-Fc and Fo-Fc maps for these last residues. The refinement statistics stall at at 26.8 and 30.6 (R and Rfree using the same test set as the low res case except extended for the higher resolution). This seems rather high considering that only 7 residues are missing. Xtriage shows an elevated wilson statistic for this dataset: Wilson ratio and moments Acentric reflections I^2/I^2:2.168 (untwinned: 2.000; perfect twin 1.500) F^2/F^2:0.772 (untwinned: 0.785; perfect twin 0.885) |E^2 - 1|:0.762 (untwinned: 0.736; perfect twin 0.541) Centric reflections I^2/I^2:3.376 (untwinned: 3.000; perfect twin 2.000) F^2/F^2:0.616 (untwinned: 0.637; perfect twin 0.785) |E^2 - 1|:1.059 (untwinned: 0.968; perfect twin 0.736) I'm particularly concerned that I^2/I^2 for both centric and acentric reflections are elevated above the untwinned case, suggesting the symmetry/space group is incorrect. R32 has C2 and R3 underneath it but reintegrating into those datasets and MR of the best model doesn't change the refinement statistics or the wilson ratio statistics, (stuck in high R/Rfree and greater than 2 and 3 for the acentric and centric reflections).This is refining with NCS or appropriate twin laws for those space groups. I would love to entertain comments. Thanks FR Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Using SAXS data for phasing at mediocre resolution.
Hi all I'm looking for anyone who has had (practical) experience using SAXS data to phase 4.2 A crystals. Please email me. FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] suggestions for SIR with strong diffraction anisotropy in the derivative
Hi all I'm trying SIR on my crystal both are C222. Native Cell Dimensions: 97.66 243.45 87.62 90 90 90 Resolution: 4.3 Derivative Cell Dimensions: 96.9100 244.66 87.61 90 90 90 Resolution: 4.7 . I plan to scale together using FHScal. Ctruncate says that my derivative has strong diffraction anisotropy. How concerned should I be about this at the level of phasing? Is there anything to be done with the anisotropy issue? Thanks - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] strange pattersons
Hi All Im receiving some strange patterns in my pattersons. Space group is C222 with no confidence (due to resolution) of systematic absences to transform to C2221. Thanks! FR pattersonmap.pdf Description: Adobe PDF document - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] a simple question: how is redundancy measured?
For a given reflection (h,k,l) how much does each situation increase the redundancy ? and which maximizes the ability to measure anomalous differences? (so assume we're separating friedel pairs here) a) measuring the same reflection again b) measuring a symmetry related reflection c) both a+b Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
